A sensitive method for quantifying mouse plasma alpha-macroglobulins (AM) using an inhibition ELISA is described. AM are important plasma proteinase inhibitors that possibly act also as immunomodulatory molecules. The standard protocol developed in our experiments involves coating well with 10 micrograms/ml A2M in carbonate buffer, followed by incubation with a 1:1 (v/v) mixture of the plasma to be tested (diluted 1/1000) and goat anti-AM (diluted 1/1250). This is followed by further incubation, first with the enzyme-conjugated antibody and with the substrate prior to the reading of absorbance levels of the reaction products. Standard curve samples must be included in each plate, employing known amounts of the purified Murine Alpha-2-Macroglobulin (MuA2M) used for coating, with concentrations ranging from 0.001 to 10 micrograms/ml. Using test samples in triplicates and a 6-point standard curve in a single ELISA plate, 25 plasma samples can be tested accurately. The method offers an useful tool for establishing AM levels in small samples of mouse plasma.
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